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Safety

Safety is of primary concern in every profession, including professions in research. Because the iGEM competition inevitably involves genetically modified organisms (GMOs), working safely is of utmost importance to its participants and their environments. The Amsterdam team has discussed the threats to safety involved with, and caused by, their work. We conclude that, while caution and proper lab procedure should be maintained, the safety of the public, the environment, our colleagues and ourselves should be in no significant danger during work on our project. However, when organisms carrying our CryoBricks are used for other applications proper care must be taken to prevent them from crossing into the environment.

The Researcher

The labs we work in are stocked with hazardous chemicals, however they are stored securely and treated with appropriate caution, as per ML-I regulations. Gloves, labcoats, masks and safety glasses are used to protect against harmful chemicals, and material safety datasheets are available in the lab.

The strain of E. coli used in our experiments (Top10) is not pathogenic. The CryoBricks we develop don't encode substances that are known or suspected to be toxic, or involved with virulence or infection. Because of this, we have deemed standard lab safety regulations sufficient for work on this project, and no additional considerations were taken into account.

Biosafety at the UvA

Even though there is no biosafety committee at the UvA for us to discuss our project with, one of our chief advisors is Dr. Pernette J. Verschure. She is tasked with overseeing biosafety at the Nuclear Organization Group (NOG) of the Swammerdam Institute for Life Sciences (SILS). She takes care of the GMO database, safety, and official registration of GMOs (i.e. GGO 01-045, 01-052 and 02-241) of the NOG. Dr. Verschure is closely involved with the project, attends all our meetings, and keeps an eye out to ensure we maintain a safe working environment.

The directives and protocols pertaining to biosafety are in care of the Dutch government and arranged on an national and international level. An overview of all national biotechnology acts may be found [http://www.overheid.nl/biotechnologie/regels/ here]. Most of the listed regulations are enforced in the entire European Union. One regulation of particular relevance with regards to safe handling of GMOs is the [http://bch.cbd.int/protocol/ Cartagena protocol], which has been signed into law by the United Nations in 2003.

Because of our previous experience in a laboratory setting, all of the teammates having at least some practical knowhow, no special training or workshops were arranged to familiarize us with safety and lab equipment. Two tours of the lab, one two weeks prior to and one on the first day of experimental work, clarified general lab procedures. The close involvement of our instructors further saw to it that we worked safely and securely.

Public

Undesirable exposure to our GMOs is unlikely because they are treated with care. Still, the possibility of our cells entering the environment exists. Since E. coli is adapted to endotherms’ intestines, our strain could thrive in such an environment. However, increased cold tolerance is not beneficial at body temperature. Even if exposure occurs, due to the non-pathogenic nature of our bacteria, there are no direct risks to the public health; only to their environment.

Environment

Efforts have been made to restrain the GMOs we work with to a controlled lab environment. All waste is handled in accordance with general safety regulations and chemicals are treated as specified by their supplier. Organic waste is disposed of in biohazard containers, autoclaved and/or treated with bleach. The windows are kept closed, and hands are washed when entering and leaving the lab.

If these precautions prove to be insufficient in preventing our GMOs from leaving the lab - for example, when accidentally flushed down the drain - they will most likely find themselves in an environment to which they are not well adapted. While the cold resistance genes we equip our bacteria with may relieve the stress caused to the cells by no longer growing in a 37°C stove, this is only one of many problems E. coli will face outside of the lab. We expect nutrient availability to be a strongly limiting factor. Furthermore, in the case of our bacteria, competition with better adapted organisms is expected to rapidly out-compete any GMOs transferred to a natural environment. However, our genes conferring cold resistance could conceivably be transferred to other bacteria through horizontal gene transfer. It is not unthinkable that an E. coli containing CryoBricks can transfer these to other bacteria via conjugation. This can grant the recipient an increased cold tolerance.

Uncontrolled spread of CryoBricks may enhance the ability of bacteria to survive in cold environments, which can have widespread consequences. For example, a bacterium that can't normally grow in a refrigerator might gain this ability by picking up a CryoBrick. This may lead to a "fridge infection”. An increased risk of fridge infections is but one of many consequences the spread of CryoBricks may have.

Precaution

Note that some of our bricks are placed under control of a non-constitutive promoter. The CryoBricks containing a derivative of the pBAD promoter will only be transcribed in presence of the sugar arabinose. This sugar is a viable carbon source for many different bacteria, and will be rapidly consumed in absence of a more preferred carbon source such as glucose. In a lab environment, glucose concentrations can be kept high. Because glucose is a more preferred carbon source, this results in catabolite repression, which prevents the consumption of arabinose, keeping the promoter activated. In natural environments, catabolite repression is more of a dynamic process and arabinose is likely to suffer quick degradation thus inactivating the promoter.

Our other CryoBricks contain the pLAC promoter, which is naturally inhibited by the lacI protein. In the TOP10 strain this promoter is constitutive, as TOP10 is a lacI-knockout strain. conversely, cells that do express the lacI protein would not constitutively express the proteins following the pLAC promoter.

Use of our biobricks

In our applications page we mention our CryoBricks show the potential of being of use to other iGEM projects, or companies.

If our bricks are used outside a lab environment, there are additional risks to take into account. For example the 2010 Delft team attempted to design an E.coli that was able to remove oil from the ocean after an oil spill. To this end. E.coli had to be modified to survive under the conditions of an ocean, such as high salt concentration. Our CryoBricks could be useful in surviving the cold temperatures found in such an environment. However, if a bacterial strain containing our biobricks is to be used in the ocean, there will be an additional risk associated with the introduction of a new species into an environment. To reduce this risk outside a lab environment a “socio-technical network” should be present. This means that all personnel handling the GMO’s are required to be qualified to do so. The organism should also be kept inside of a closed system, in which it is separated from the environment. If no malfunctions occur, this would be enough to prevent an environmental outbreak of the organism with our biobricks.

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